Gene trapping in mouse embryonic stem cells offers a powerful tool to create insertional mutations in mice that are immediately accessible to molecular analysis. However, certain deficiencies in the technology have been recognized and need to be addressed before embarking on a large-scale effort to trap each of the 30,000 or so genes in the mammalian genome. These drawbacks include the incompatibility of the mouse strains used for gene trapping (and gene targeting) in relation to classical ENU mutagenesis efforts now underway. More importantly, the versatility of conventional gene trapping approaches is limited and does not allow for the generation of other, more useful, alleles that are needed to fully address gene function. The goal of this grant is to develop optimal gene trap vector designs and methods for the large-scale functional analysis of genes in mice. Specifically, this proposal aims to isolate highly germline-competent, feeder-independent embryonic stem cells from the C57BL/6 strain of mice, to construct "universal" gene trap vectors capable of targeting all classes of genes, and to design and test the feasibility of gene trap vectors with site-specific recombination sites. The strategic placement of site-specific recombination sites will permit secondary modifications at the gene trap locus enabling the creation an unlimited variety of desirable alleles. These modifications include, but are not limited to, 1) the generation of an allelic series of mutations in the trapped gene (null, hypomorphic, and site-specific mutations), 2) the creation of conditional alleles (temporal and tissue-specific), 3) the inclusion of reporter genes for the study of gene expression and cellular functions (Bgal, GFP, PLAP, etc.), 4) the introduction and controlled expression of recombinases (Cre, fip, etc.) and transactivators (TetR, gal4, etc.), 5) the production of knock-ins of heterologous gene products, and 6) the addition of negative drug selection cassettes (HPRT, TK) for engineering chromosomal deletions and inversions. The technology described in this proposal will have wide applications to all areas of mammalian biology and will complement and extend other mutagenesis strategies in mice.